1. Field of the Invention
The present invention relates to a probe carrier such as what is called “nucleic acid chip” having a plurality of nucleic acid probe immobilized areas arranged on a carrier such as a substrate in a matrix form. The present invention also relates to a method for analyzing an amount of nucleic acid probes in each dot-like immobilized area formed on a probe carrier by time-of-flight secondary ion mass spectrometry (hereinafter abbreviated as TOF-SIMS).
2. Description of Related Art
A nucleic acid chip such as a DNA chip or RNA chip is now being used to acquire genetic information as in the analysis of a genome or the analysis of gene expression. The results of analysis using those chips are expected to provide important indices for diagnoses of cancer, hereditary diseases, lifestyle-related diseases, infectious diseases, and the like, and prognosis, determination of therapeutic strategy, and the like.
There are known several methods of producing the above nucleic acid chips. Taking a DNA chip as an example, typical DNA chip producing methods include one in which photolithography is used to synthesize DNA probes on a substrate sequentially (U.S. Pat. No. 5,405,783) and one in which pre-synthesized DNA or complementary DNA (cDNA) is supplied and bonded to a substrate (U.S. Pat. No. 5,601,980), JP H11-187900 A, “Science” Vol. 270, 467, 1995, etc.).
In general, the nucleic acid chip is produced by one of the above methods. When the nucleic acid chip is to be used for the above purposes, it is very important to know the amount of probes existent on each matrix, that is, density in order to ensure reliable analysis, i.e., determination and reproducibility. It is also important to know (imaging) what matrix form (shape, size and state) the probes are actually existent in. However, since the probes on the chip are existent in the form of a monomolecular film theoretically, a highly sensitive surface-analysis technique is required for the analysis of the probes.
As the highly sensitive surface-analysis technique, a method for labeling a probe with an isotope is known. However, since this method is complicated and dangerous and requires a special device and facility, it is not generally used in many cases.
Alternative methods include a method for labeling probes by fluorescence and a method for bonding a substance labeled with fluorescence to probes, that is, fluorescence hybridization. However, these methods involve problems with stability of a fluorochrome, quenching, non-specific adsorption of fluorochrome to the surface of a substrate and the determination (stability, reproducibility) of specific bonding (hybridization). Consequently, there are still remaining problems to be solved for the determination of the amount of probes themselves in many cases.
Alternatively, general highly sensitive surface-analysis methods include ATR making use of FT-IR (Fourier transform infrared spectroscopy) and XPS (X-ray photoelectron spectroscopy). However, it cannot be said that those methods attain sufficient sensitivity for the quantitative analysis of the probes of the nucleic acid chip or imaging. In particular, when general glass is used in the substrate of the nucleic acid chip, FT-IR (ATR) has a problem such as the influence of absorption by glass and XPS has a problem such as the influence of a charge-up, for example. Therefore, they may not be effective analysis methods in some cases.
Other highly sensitive surface-analysis methods include a DNA detection method making use of laser resonance ionization spectroscopy (RIS) as disclosed in U.S. Pat. No. 5,821,060. In this method, a laser beam having a wavelength corresponding to the ionization energy of an element of interest released from the surface of a sample is applied to ionize the element and detect this ionized element. As a method for releasing the element from the surface of the sample, a method using a laser beam is disclosed. However, this method has such problems that a large-size apparatus is required and that the element to be detected is limited.
As still another highly sensitive surface-analysis method is dynamic secondary ion mass spectrometry (dynamic-SIMS) which is not suitable for the analysis of an organic material such as a nucleic acid-related substance because an organic compound is decomposed into small fragment ions or particles during the generation of secondary ions and thereby chemical structure information obtained from its mass spectrum is poor.
On the other hand, there is known time-of-flight secondary ion mass spectrometry (TOF-SIMS) as highly sensitive surface-analysis method. TOF-SIMS is an analysis method for investigating what type of atom or molecule is existent on the outermost surface of a solid sample and has the following features.
That is, it can detect a trace component even when its amount is as small as 109 atoms/cm2 (corresponding to 1/105 of a one-atomic layer on the outermost surface), can be used for both organic and inorganic materials, can measure all the elements and compounds existent on the surface and can effect imaging of secondary ions from a substance existent on the surface of the sample.
The principle of this method will be briefly described hereinbelow. When a pulse ion (primary ion) beam with high energy is applied to the surface of the solid sample under high vacuum, a constituent of the surface is released into vacuum by a sputtering phenomenon. Positively or negatively charged ions (secondary ions) generated at this point are converged in one direction by an electric field and detected at a fixed distance. When primary ions are applied to the surface of the solid sample in a pulse form, secondary ions which differ in mass are generated according to a composition of the surface of the sample. Since light ions fly faster than heavy ions, the mass of each of the generated secondary ions can be analyzed by measuring the time (time of flight) from the generation of the secondary ions to the detection of the ions. When the primary ions are applied, only secondary ions generated from the outermost side of the surface of the solid sample are released into vacuum, thereby making it possible to obtain information on the outermost surface (depth of about several Å to several nm) of the sample. Since the amount of the primary ions applied is extremely small in TOF-SIMS, the organic compound is ionized while it keeps its chemical structure, so that the structure of the organic compound can be known from its mass spectrum. For an insulating sample, a pulsed electron beam of low energy is applied to the positively charged surface of the sample when the pulsed primary ions are not irradiated in order to neutralize the positive charges accumulated on the surface of the solid sample, thereby making it possible to analyze the insulating sample. In addition, TOF-SIMS gives an ion image (mapping) of the surface of the sample to be measured by scanning a primary ion beam.
An example in which the nucleic acid in the form of a monomolecular film immobilized on the substrate is detected by TOF-SIMS has been already reported (Proceeding of the 12th International Conference on Secondary Ion Mass Spectrometry 951, 1999). In this example, the decomposed fragment ions of a base and the decomposed fragment ions of a phosphate backbone are enumerated as nucleic acid fragment ions detectable by TOF-SIMS.
As attempts to carry out quantitative analysis using TOF-SIMS, there are known one in which standard solutions having different concentrations are each applied to a clean silicon substrate, dried and measured by TOF-SIMS to obtain an analytical curve from a peak intensity of secondary ions from the resulting standard samples and compare it with the peak intensity of secondary ions from a sample to be analyzed (C. M. John et al., SIMS VIII, p. 657, Wiley and Sons, 1992) and one in which a standard sample for total reflection fluorescence X-ray analysis prepared by spin-coating a silicon substrate with a trace amount of a metal element to is used (P. Lazzeri et al., Surface and Interface Analysis, Vol. 29, 798 (2000)).
However, quantitative analysis by TOF-SIMS involves the following problems. That is, in the method reported by C. M. John et al. in which standard solutions having different concentrations are each applied to a clean silicon substrate, dried and measured by TOF-SIMS to obtain an analytical curve from the peak intensity of secondary ions from the resulting standard samples and compare it with the peak intensity of secondary ions from a sample to be analyzed, hydrocarbon and the like (contamination) are deposited on the prepared standard sample formed on the silicon substrate with time or the standard substance itself undergoes a chemical change, thereby losing reliability when the same standard sample is continuously used. For the above reasons, the standard solution must be prepared each time to improve determination accuracy, thereby making an operation troublesome.
The method reported by P. Lazzeri et al. in which a standard sample for total reflection fluorescence X-ray analysis prepared by spin-coating a silicon substrate with a trace amount of a metal element is used lacks reliability because the above surface contamination and oxidation occur when the standard sample is kept for a long time, and is not always suitable for measurement by TOF-SIMS in which a size of the area to be analyzed is several tens of μm to several hundreds of μm due to nonuniform distribution of the elements of interest when spin-coating is used. Further, the standard sample for total reflection fluorescence X-ray analysis has conductivity, whereas a nucleic acid chip may be formed on an insulating material substrate. In this case, TOF-SIMS measurement conditions which are determined by the standard sample on a silicon substrate may not always be measurement conditions for a nucleic acid chip, thereby causing an error due to differences in measurement conditions.